As life science research advances, it becomes well-recognized that nucleic acid is the key substance for the determination of genetic information. By determining changes or mutations in the nucleic acid sequences in a sample of a subject, one can determine whether the subject carries pathogenic microbes and/or resistance to such microbes, whether the subject has certain diseases, and whether the subject is under certain genetic state. Therefore, nucleic acid analysis techniques find application in various areas of life science research, including testing, classification, and detection of drug resistance genes of pathogenic microbes, diagnosis and prognosis of diseases, HLA classification, and SNP detection.
Because the amount of nucleic acid in a sample is usually insufficient for analysis, it is usually necessary to amplify the nucleic acid to be detected prior to the analysis. Methods of amplification include polymeric chain reactions (PCR), reverse transcription polymeric chain reaction (RT-PCR), strand displacement amplification (RDA), and rolling circle amplification (RCA). Andras et al., Mol. Biotechnol., 19:29-44, 2001. Among those, PCR is currently used most often. There are many methods for analyzing PCR products. For example, agarose gel electrophoresis or PAGE electrophoresis have been used for detection of PCR products. These electrophoresis methods provide fast and convenience analyses. However, these methods suffer from low specificity and are thus unsuitable for gene mutation analyses. Another method for analyzing PCR products is use of restriction endonuclease, which has limited application, low sensitivity, and is hard to operate. One method that ensures accuracy in sequence information of PCR product involves cloning the PCR product and sequencing the cloned sequence. That method, however, involves multiple steps and is thus costly and non-practical.
Methods for hybridization of PCR products with probes include: 1) Southern hybridization, i.e., use electrophoresis to separate out the PCR products, transfer the PCR product to a membrane, and hybridize the PCR products with a labeled probe. This technology produces good specificity, but is complicated in operation, time consuming, and thus not suitable for parallel analysis of multiple features. 2) Positive dot hybridization, i.e., immobilize PCR products on the surfaces of a membrane or other solid substrates and hybridize the immobilized PCR products with a probe. This method requires that the PCR products be purified, quantitated, and immobilized, and is time consuming and unsuitable for detection in small amounts of samples. 3) reverse dot hybridization, i.e., hybridize the PCR products to probes that are previously immobilized to the surface of a membrane or other solid substrates. A substrate containing a large number of probes can be prepared ahead of time, and can be used to analyze PCR products immediately after the completion of the PCR reactions. The method is thus fast and convenient, and is suitable for use in kits and gene chips.
Gene chip technology is revolutionary. Due to its systematic, microdized, and automatic characteristics, gene chip technology finds important applications in nucleic acid analyses, particularly in high throughput nucleic acid analyses. Debouck and Goodfellow, Nature Genetics, 1999, 21 (Suppl.):48-50; Duggan et al., Nature Genetics, 1999, 21 (Suppl.):10-14; Gerhold et al., Trends Biochem. Sci., 1999, 24:168-173; and Alizadeh et al., Nature, 2000, 403:503-511. Nucleic acid chips have been used to analyze gene expression profiles under specific conditions, and have also been used to determine single nucleotide polymorphism (SNP) in gene regions that are up to 1 kb. Guo et al., Genome Res., 2002, 12:447-57.
Traditional passive nucleic acid analysis using biochips (for example biochips used for detection of infectious diseases) typically include three separate steps. The first step is sample preparation, i.e., preparation of nucleic acids from samples such as plasma, blood, saliva, urine, and feces. The nucleic acids obtained from such samples are usually insufficient to be analyzed directly, and need further amplification, such as PCR amplification. The second step is nucleic acid hybridization, i.e., hybridization between the amplified product and the probes immobilized on the chip. The third step is detection of hybridization signals, which is typically carried out by detection of certain labels, which can be introduced during the process of amplification and hybridization. The method of detection depends on the labels that are used. For example, fluorescence detector can be used to detect fluorescent labels, while autoradiograms can be used to detect radiolabels. In cases where biotin and straptavidin labels are used, further enzymatic amplification can be carried out. Different amplification methods are used depending on the desired sensitivity of the experiment. For example, Tyramide signal amplification (TSA) and branched DNA methods as described in Karsten et al., Nucleic Acids Res., 2002, E4 and Kricka, Clin. Chem., 1999, 45:453-458, respectively.
Hybridization between target nucleic acid and probes immobilized on the surface of the biochip constitutes a central step in the nucleic acid detection. The target nucleic acid is typically amplified by PCR, denatured into single-chain PCR products, which are in turn hybridized to probes under stringent conditions. The hybridized product is then washed and detected. During hybridization, only one chain of the PCR product can hybridize with the probe. The corresponding complement chain may interfere with the hybridization due to self-annealing of the PCR double chain product. As a result, hybridization signals may be lost. It has been found that, when hybridized to an oligonucleotide probe, the hybridization sensitivity of a single chain DNA is about five times higher than that of denatured double-stranded DNA. Kawai et al., Anal. Biochem. 1993, 209:63-69. Thus, it is desirable to obtain single chain nucleic acid for high efficiency hybridization with oligonucleotide probes on gene chips.
There are several methods of preparing for single chain nucleic acid. In addition to denaturation of double stranded DNA by heat or base, methods of preparing single chain nucleic acid include the following.
1. The reverse transcription method. In this method, a T7 promoter is added to a PCR primer. A single chain nucleic acid is produced by T7 RNA-polymerase-mediated in vitro transcription using purified PCR product as a template. Hughes et al., Nat. Biotechnol., 2001, 19:342-347. Although the yield of single chain nucleic acid is quite high with this method, such two-step method is inconvenient and prone to contamination by RNAase.
2. Exonuclease cleavage method, Higuchi and Ochman, Nucleic Acid Res., 1989, 17:5865. In this method, one of the PCR primers is phosphorylated. When the PCR product is subject to cleavage by an exonuclease, the chain that extends from the phosphorylated primer would not be cleaved. The exonuclease will then have to be heat inactivated. This method requires purification of PCR products and relies on exonuclease activity, and is thus inconvenient.
4. Denaturing high-performance liquid chromatography (DHPLC). In this method, one of the PCR primers is labeled with biotin. The chain that extends from the labeled primer can therefore be separated from the other chain in DHPLC. Dickman and Hornby, Anal. Biochem., 2000, 284:164-167. The desired single chain can be obtained from the double stranded PCR product within 15 minutes. Such method, however, requires expensive machinery and thus cannot be commonly used.
4. Magnetic bead capturing method. In this method, biotin is attached to one of the PCR primers. The chain that extends from the labeled primer can be captured by straptavidin-coated magnetic beads, and dissociated from the beads using NaOH. Espelund, et al., Nucleic Acids Res., 1990, 18:6157-6158. This method is very expensive due to the use of coated magnetic beads.
5. Asymmetric PCR. While all the above methods involve extra steps after the PCR reactions, asymmetric PCR allows preparation of DNA during the PCR reaction process. We found that both asymmetric PCR and magnetic bead capturing method produce relatively high sensitivity and specificity, while heat denaturation and base denaturation methods often produce false negative results. Among all these methods, asymmetric PCR is relatively simple and low cost, and is thus much more practical. Gao et al., Analytical Letters, 2003, 33:2849-2863.
Currently, there are the following schemes for asymmetric PCR.
1) Use different concentrations of upstream and downstream primers for asymmetric PCR. As the cycles increase, primers with a lower concentration are used up, while primers with a high concentration continue to produce single chain DNA at a linearly increasing rate. Gyllensten and Erlich, Proc. Natl. Acad. Sci. U.S.A., 1988, 85:7652-7656. Similarly, Zihong He et al. used primers at a 1:10 and 1:20 ratio to asymmetrically amplify IGF-II genes for SNP detections. He et al., Chinese Journal of Sports Medicine, 2002, 21:116-121. Shuangding Li et al. used primers at ratio of 1:15 to asymmetrically amplify HLA-DRB1 gene. Li et al., Chinese Journal of Experimental Hematology, 2003, 11:393-397. Such method requires optimization of the ratio between upstream and downstream primers, and increases possibility of nonspecific amplification. As a result, the product is usually shown as a diffused band on electrophoresis. Erdogan et al., Nucleic Acids Res., 2001, 29:E36.
2) Use different lengths of upstream and downstream primers for asymmetric PCR. Xiaomou Peng et al. used a 34-nucleotide upstream primer and a 20-nucleotide downstream primer to asymmetrically amplify the S gene of HBV. During the second phase of the PCR cycles, the annealing temperature is increased. The short primers are unable to anneal under such conditions, while the long primers continue to extend to obtain single chain nucleic acid. Peng et al., Chinese Experimental Diagnostics, 2002, 6:206-208. Although the primers are gene-specific, the use of long primers introduces nonspecificity in amplification. Such method is therefore not suitable for the amplification of genes with many SNP sites (such as bacterial 16S rRNA gene).
3) Use of symmetric PCR to generate a PCR template, and use a single primer or an unequal amount of to further asymmetrically amplify and label the PCR product. Gorelov et al., Biochem. Biophys. Res. Commun., 1994, 200:365-369; Scott et al., Lett. Appl. Microbiol., 1998, 27:39-44; Guo et al., Genome Res., 2002, 12:447-457; Zhou et al., Medical Animal Control, 2003, 19:524-527. These methods use purified symmetric PCR product as a template, and subsequently use single primer to produce single chain via PCR cycles. The method requires multiple steps of reactions, and is therefore time consuming and inconvenient.
Traditional methods of multiplex PCR require multiple levels of optimizations. These methods have the following problems. 1) The use of multiple primers generates false positive amplification by these different primers. 2) Competition among the different primers create unbalanced amplification of the target nucleic acids, that is, certain primer pairs amplify efficiently while certain primer pairs amplify very inefficiently. 3) It is different to repeat the experiment. It therefore will be even more unsatisfactory to further carry out asymmetric PCR under such circumstances. The methods described above are therefore not suitable for the simultaneous analysis of multiple targets. There is a need for single-step asymmetric PCR.